NextSeq 550 sequencing; RNA-seq of Tfh cells from BALB/c and C57Bl/6 mice immunized with OVA-IFA or OVA-CpG
RNA-seq of Tfh cells from BALB/c and C57Bl/6 mice immunized with OVA-IFA or OVA-CpG
Mice aged between 8 to 12 weeks were immunized subcutaneously in the footpad with ovalbumin (Ovalbumin EndoFit, Invivogen, #vac-pova) either emulsified 1:1 (v:v) with incomplete Freund's adjuvant (IFA, Sigma-Aldrich, #F5506), admixed with CpG (ODN 1826 - TLR9 ligand, Invivogen, #tlrl-1826), or entrapped with CpG in polymeric nanoparticles (nano-CpG). Each animal was inoculated in the paw with one of the antigen/adjuvant mixtures, with a volume of 50 μL per paw containing 80 μg of OVA and, in the case of CpG or NP-CpG formulations, 30 μg of CpG. Popliteal lymph nodes were collected on day 11 following immunization. Single-cell suspensions were obtained by disrupting the lymph nodes in PBS (Lonza) with 2% FBS (Gibco) with curved forceps and a nylon mesh. Surface stainings were performed in the same buffer and a viability dye was used for dead-cell exclusion. Cells were sorted on a BD FACSAria cell sorter directly into DNA LoBind tubes (Eppendorf, #0030108051) containing nuclease-free water (Sigma-Aldrich, #W4502) with 0.2% Triton X-100 (Sigma-Aldrich, #T8787) and RNAse inhibitor at 2 U/mL (RNaseOUT, Invitrogen, #10777019). Cells were frozen in dry ice and kept at -80 ºC until further processing. There was no RNA isolation, the cDNA was prepared directly from the cell lysate. cDNA preparation was done directly from cell lysatesat GeneCore EMBL facility, according to the protocol described by Picelli et al. for Smart-seq2 (Nat Protoc. 2014 Jan;9(1):171-81. doi: 10.1038/nprot.2014.006), and sequencing libraries were prepared based on the tagmentation protocol described by Hennig et al. (G3 (Bethesda). 2018 Jan 4;8(1):79-89. doi: 10.1534/g3.117.300257).